Project Summary One in twenty-six Americans will develop epilepsy during their lifetime. Unfortunately, one-third of epilepsy patients will not achieve seizure freedom with conventional therapies. These patients are at greatest risk for sudden unexpected death in epilepsy (SUDEP), the leading cause of death in patients with refractory epilepsy. While the exact etiology of SUDEP is unknown, it is thought that cardiorespiratory dysfunction and arousal impairment are involved. Suppression of EEG activity following a seizure, or post-ictal generalized EEG suppression (PGES), may correlate with SUDEP risk. The origin of PGES is unknown, but during PGES patients experience stupor and unresponsiveness. Serotonin (5-HT) is broadly implicated in SUDEP due to its role breathing, sleep/wakefulness, and arousal. The dorsal raphe nucleus (DRN) is a key source of 5-HT projections. DRN 5-HT activity is depressed by seizures. We hypothesize PGES may represent an electrographic marker of impaired arousal consequent to seizure-induced DRN dysregulation. Our preliminary data in mice indicate that systemic application of a selective serotonin reuptake inhibitor (SSRI) or direct chemical or optogenetic stimulation of DRN 5-HT neurons prior to an induced seizure shortens PGES duration. However, the specific network and receptor mechanisms underlying PGES are unknown. Our objective is to identify a DRN network and 5-HT receptor mechanism that could be manipulated to reduce PGES and prevent SUDEP. A potential downstream target is the pedunculopontine tegmental nucleus (PPT), a pontine region involved in sleep-wake regulation, attention, EEG regulation, and arousal. Seizure-induced dysregulation of 5- HT signaling may interfere with subcortical arousal networks, such as those involving the PPT, and produce PGES. In Aim 1, to determine a role for a DRN ? PPT circuit in PGES, we will optogenetically stimulate and inhibit DRN 5-HT terminals in the PPT prior to seizures induced by amygdala stimulation in amygdala kindled mice during wake/NREM/REM and observe changes in PGES duration. Several 5-HT receptors have been implicated in SUDEP and may be found on cholinergic neurons in the PPT. In Aim 2, we will utilize immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, RNAscope fluorescent in situ hybridization, and immunohistochemistry to determine the identity of PPT neurons and 5-HT receptors contacted by DRN 5-HT terminals. Then we will administer intracranial 5-HT antagonists into the PPT with or without a selective serotonin reuptake inhibitory onboard, induce an amygdala-kindled seizure, and observe effects on PGES. Participation in the proposed training plan and completion of the proposed experiments will advance the applicant?s neuroscience training. It will also elucidate the DRN-PPT circuit and determine its role in PGES. By manipulating 5-HT circuitry, we may discover ways to eliminate PGES and consequentially prevent death in high risk epilepsy patients.